Environmental Hot Spots and Resistance-Associated Application Practices for Azole-Resistant Aspergillus fumigatus, Denmark, 2020–2023

Azole-resistant Aspergillus fumigatus (ARAf) fungi have been found inconsistently in the environment in Denmark since 2010. During 2018–2020, nationwide surveillance of clinical A. fumigatus fungi reported environmental TR34/L98H or TR46/Y121F/T289A resistance mutations in 3.6% of isolates, prompting environmental sampling for ARAf and azole fungicides and investigation for selection of ARAf in field and microcosmos experiments. ARAf was ubiquitous (20% of 366 samples; 16% TR34/L98H- and 4% TR46/Y121F/T289A-related mechanisms), constituting 4.2% of 4,538 A. fumigatus isolates. The highest proportions were in flower- and compost-related samples but were not correlated with azole-fungicide application concentrations. Genotyping showed clustering of tandem repeat–related ARAf and overlaps with clinical isolates in Denmark. A. fumigatus fungi grew poorly in the field experiment with no postapplication change in ARAf proportions. However, in microcosmos experiments, a sustained complete (tebuconazole) or partial (prothioconazole) inhibition against wild-type A. fumigatus but not ARAf indicated that, under some conditions, azole fungicides may favor growth of ARAf in soil.


Air
August 2021 38 divided between two localities Air samples were collected using a Sartorius air sampler, with subsequent application of exposed gelatine filtres directly on agar culture plates.Sampling was done both before, during and after harvesting at two localities (Flakkebjerg, Dannemare).Fields were treated with standard fungicides including prothioconazole and tebuconazole.No soil samples were taken from the fields where air samples were collected.Air samples were not applicable for azole concentration determination.Soil Summer 2022 5 sampling times from 4 treatments x 2 locations = 40 samples Samples were taken from two wheat trial sites (Fredericia (Jutland), Flakkebjerg (Zealand)) where two treatments with different azoles (prothioconazole, tebuconazole, mefentrifluconazole) were been applied twice (Table 7).Both untreated and treated plots were sampled, before spraying, before the 2nd spray and after 3, 6 and 10 weeks after the 2nd spray.Each sample included soil from 4 replicates.The Flakkebjerg site was further treated with 0,25 Folicur 250 EC (tebuconazole) to reduce a risk of yellow rust, which unintededly developed in the wheat trial, which had focus regarding control of Septoria tritici blotch.Flower beds & Flower producers Soils April 24 th and May 17 th 2021 4 parks x 3 samples (2 x T, 1 x K, 1 x F) 2 gardens x 3 samples Samples were taken at three flower beds per park/garden.Three public parks North Zealand and near Copenhagen) and two private gardens (Flakkebjerg and Copenhagen) were sampled.Samples were all taken in connection with spring flowers (tulips and other bulb plants).
Sample type Date N. of samples Comments =18 samples None of the parks nor gardens had any history of azole fungicides application in the recent history.

Soil
Nov 9 th 2020 2 x 10 pots with poinsettia 1 x 10 cactus 1 x 10 campanula = 40 samples Samples were taken from the greenhouses, growing flower pots (Table 8).Sampling was done just before the plants were ready to be sold.Potted plants had all been treated with either metconazole or paclobutrazole used as a plant growth regulator.Pot topsoil (top 5 cm) from 4 different cultures from Fyn were analysed.Soil 2021 10 samples of poinsettia One of the flower growers from 2020 was revisited and poinsettia was resampled.Top soil from flower pots was analysed.Air samples were also taken from the same grower's greenhouses.a PCR conditions for both assays were as follows; 1x Quanta ToughMix® (dNTPs, polymerase, buffer and "low ROX", QuantaBio distributed by VWR, Søborg, Denmark), 0.5 µM forward and reverse primer, 0.4 µM probe for assay (1) and 0.33 µM probe for assay (2), 0.8 mM and 3.5 mM additional MgCl2 for assay (1) and (2), respectively, 5 µL DNA and molecular biology grade Gibco water (Thermo Fisher Scientific, Roskilde, Denmark) in 30 µL reaction volumes.All PCR reactions were run on the Quantstudio 5, thermocycler (Thermo Fisher Scientific, Roskilde, Denmark) with 5 min denaturation at 95 oC followed by 45 cycles of denaturation (20 s at 95 oC) and annealing (1 min at 60 oC) with data acquisition at this step and ramp rates of 1.6 oC/s.PCR data was analysed with the Quantstudio Design & Analysis Desktop software v1.5.2 (Applied Biosystems by Thermo Fisher Scientific, Denmark).Sensitivity was evaluated by 10-fold dilution series of normalised DNA concentrations and spore suspensions, extracted on Nuclisens easyMag (biomérieux, Denmark), using 1 mL input volume of supernatant from bead-beated spore suspensions and 60 µL elution volume.The limit of detection (LOD) was estimated to 50 fg DNA (<20 copies) and 50-200 CFU/mL, respectively with no differences between the three probes (A.fumigatus cyp51A, TR34 and TR46) (data not shown).*Previously, this probe was labelled with ABY fluorescence dye to enable triplexing with the CYP-P3 and the TR34-P3 probes and to enable detection of all three in a single PCR reaction.The results, however, indicated cross-reaction and undesired interference of fluorescence, and thus for the majority of the samples the TR46-probe was either run as duplex with the CYP-P3 FAM probe or alone.

A. fumigatus quantification in microcosmos experiments:
For the first experiments, the target was a multicopy internal transcribed spacer (ITS, Assay ( 1)).Ct value to CFU/g soil conversion was based on a standard curve with known CFU and Ct values (log(X)=(Y-34,137)/-3,5178), where "X" = CFU/PCR reaction and "Y" = Ct value (data not shown).

*Appendix Figure 1 .
Heat map colouring is used to highlight higher concentrations of azole fungicides and those close to or higher than a tenth of the MIC for A. fumigatus are indicated in bold font.TBZ=tebuconazole, PPZ=propiconazole, MTZ=metconazole, EPZ=epoxiconazole, DFZ=difenoconazole, PTZ-S =prothioconazole-desthio, PBZ=paclobutrazole, MFZ=mefentrifluconazole. Test of growth conditions in microcosmos settings.The growth of A. fumigatus wildtype (WT) and TR34/L98H (TR34) were measured by CFU counting.Samples incubated at 20°C were followed by weekly CFU counting for four weeks and two different inoculum sizes (10 2 and 10 3 CFU) were investigated.Samples incubated at 10°C or 15°C were only investigated three weeks after inoculation with 10 3 CFU, at which time no growth was found in samples incubated at 10 °C.

Table 3 .
LOD and LOQ were determined as 3-times (LOD) and 10-times (LOQ) standard deviation of a control sample spiked at a low concentration.Empty cells indicate no surrogate standard available.PCR and sequencing primers used for cyp51A sequencing of azole-resistant A. fumigatus isolates a

Table 7 .
Primers and probes for A. fumigatus quantification PCR assays a